Efflux transporters are proteins that span the plasma membrane of a cell and catalyze the export of compounds from these cells at the expense of ATP. The first ABC transporter described was multidrug resistance protein 1 (MDR1 in human, Mdr1a/b in rodents, or P-glycoprotein; Juliano et al. Biochimica et biophysica acta 1976 455(1):152-162). This 170 kilodalton transmembrane protein, encoded by the gene name ABCB1, spans the plasma membrane six times in two distinct regions (Van der Bliek et al. Cancer Res 1988 48(21):5927-5932). When this protein was first characterized, it was discovered that cells overexpressing Mdr1a/b were resistant to colchicine, doxorubicin, and actinomycin D (Devault, A & Gros, P Molecular and cellular biology 1990 10(4):1652-1663). Following the discovery of MDR1/Mdr1, it was realized that efflux pumps exist that desensitize cells to cancer chemotherapy; however, there are many cancers that do not overexpress this ATPase (Klaassen et al. Pharmacol Rev 2010 62(1):1-96). Because of this resistance of many tumors to chemotherapy, the discovery of additional xenobiotic efflux pumps was pursued, leading to the discovery and characterization of multidrug resistance-associated protein 1 (MRP1/Mrp1; Cole et al. Science 1992 258(5088):1650-1654). Soon thereafter, MRP1 was linked to resistance to anticancer drugs (Stride et al. Mol Pharmacol 1997 52(3):344-353; Grant et al. Cancer Res 1994 54(2):357-361; and Barrand et al. Journal of the National Cancer Institute 1994 86(2):110-117).
MRP1/Mrp1 is known to transport glutathione conjugates of nitrogen mustard-derived compounds chlorambucil and melphalan (Barnouin et al. British journal of cancer 1998 77(2):201-209; Jedlitschky et al. Cancer research 1996 56(5):988-994; and Paumi et al. The Journal of biological chemistry 2001 276(11):7952-7956). Other ligands for MRP1/Mrp1 include oxidized glutathione (Minich et al. Journal of neurochemistry 2006 97(2):373-384), the topoisomerase II inhibitors doxorubicin, idarubicin (Smeets et al. Leukemia 1999 13(9):1390-1398), and etoposide (Lorico et al. Cancer research 1995 55(19):4352-4360; Tasaki et al. The Journal of urology 1995 154(3):1210-1216; Brock et al. Cancer research 1995 55(3):459-462; Abe et al. International journal of cancer Journal international du cancer 1994 58(6):860-864; Kubota et al. Cancer chemotherapy and pharmacology 1994 34(3):183-190; Schneider et al. Cancer research 1994 54(1):152-158; Hamaguchi et al. Cancer research 1993 53(21):5225-5232; Godinot et al. Molecular cancer therapeutics 2003 2(3):307-316), the lipid peroxidation product glutathione-conjuguated 4-hydroxynonenal (Renes et al. The Biochemical journal 2000 350 Pt 2:555-561; Sultana R, & Butterfield D A Neurochemical research 2004 29(12):2215-2220), glutathione-conjugated aflatoxin B1, estradiol-17β-glucoronide (Qian et al. The Journal of biological chemistry 2001 276(9):6404-6411), and glutathione-conjugated methylmercury (Klaassen et al. Pharmacol Rev 2010 62(1):1-96; Rush et al. Neurotoxicology 2012 33(3):476-481; and Suzuki et al. Seminars in liver disease 1998 18(4):359-376). Some heavy metals have also been shown to act as substrates for human MRP1, including antimony salts (Gayet et al. FEBS letters 2006 580(30):6891-6897; Mookerjee et al. Antimicrobial agents and chemotherapy 2008 52(3):1080-1093), the mercuric ion (Aleo et al. Toxicology 2005 206(1):137-151), arsenate, and arsenite (Vernhet et al. Toxicology 2000 142(2):127-134; Leslie et al. The Journal of biological chemistry 2004 279(31):32700-32708). Even for compounds transported by MRP1/Mrp1 that are not conjugated to glutathione, co-transport of glutathione, or in some cases, S-methyl glutathione (Rothnie et al. The Journal of biological chemistry 2006, 281(20):13906-13914), is still required for MRP1/Mrp1 to function (Leslie et al. The Journal of biological chemistry 2001, 276(30):27846-27854). Nine human MRPs have been characterized; an additional MRP transporter, MRP2/Mrp2 is also known to mediate extracellular transport of glutathione-conjugated electrophiles (Klaassen C D & Aleksunes L M Pharmacol Rev 2010, 62(1):1-96). MRP2, like MRP1, has been reported to transport glutathione-conjugated chlorambucil, although less efficiently than MRP1 (Smitherman et al. The Journal of pharmacology and experimental therapeutics 2004, 308(1):260-267); it is also known to efflux cisplatin, a crosslinking agent with a generally similar molecular mechanism of action as the nitrogen mustard HN2 when cisplatin is conjugated to glutathione (Wen et al. Am J Pathol 2014, 184(5):1299-1308).
The human MRP1 gene was first isolated from a doxorubicin-resistant small-cell lung carcinoma, and it was determined to be a member of the ATP-binding cassette family based on its primary sequence (Cole et al. Science 1992, 258(5088):1650-1654). This transmembrane protein is overexpressed in multidrug resistant cervical cancer HeLa cells and non-small cell lung carcinoma cell lines (Cole et al. Science 1992, 258(5088):1650-1654). It has also been demonstrated that MRP1 was overexpressed in a number of human tumor cell lines that do not express MDR1 but still possess the “multidrug resistant” phenotype. Two glioma cell lines, IN500 and T98G, have elevated MRP1 expression and are resistant to etoposide, vincristine, and doxorubicin and have a decreased accumulation of etoposide following treatment (Abe et al. International journal of cancer Journal international du cancer 1994, 58(6):860-864; Benyahia et al. Journal of neuro-oncology 2004, 66(1-2):65-70). Various inhibitors of MRP1 activity were shown to reverse resistance to etoposide and doxorubicin in human glioma cells (Abe et al. British journal of cancer 1995, 72(2):418-423). Clinically, high-grade gliomas have been demonstrated to express more MRP1 than those of a lower grade (de Faria G P et al. Cancer investigation 2008, 26(9):883-889). Research has shown a correlation between MRP1 expression and a poor prognosis for patients with breast cancer (Filipits et al. Anticancer research 1999, 19(6B):5043-5049; Abaan et al. Cancer investigation 2009, 27(2):201-205), ovarian cancer (Faggad et al. Histopathology 2009, 54(6):657-666), and neuroblastoma [52-55]. Neuroblastoma cells were also shown to have an increased MRP1 expression compared to healthy cells (Peaston et al. British journal of cancer 2001, 85(10):1564-1571). A correlation was also shown between MRP1 expression and a poor response to treatment chemotherapy in acute lymphocytic leukemia, though this study combined several different treatment regimens, some of which included MRP1/Mrp1 substrates and some of which did not (Styczynski et al. Journal of cancer research and clinical oncology 2007, 133(11):875-893). A further study of acute lymphocytic lymphoma examined patients treated with the BFM-95 protocol that includes MRP1/Mrp1 ligands methotrexate, vincristine, daunorubicin, and cyclophosphamide, and a statistically significant correlation was found between MRP1 expression and poor response to therapy (Kourti et al. International journal of hematology 2007, 86(2):166-173). Clinical research also showed that MRP1, as well as MDR1, MRP2, and MRP3 are elevated in residual tumors following treatment with MRP1 substrate doxorubicin compared with the untreated primary tumors (Tada et al. International journal of cancer Journal international du cancer 2002, 98(4):630-635).
It has been suggested, as well as observed, that multiple drugs are more effective than just one compound when treating tumors. In fact, treatment with etoposide and cisplatin or chlorambucil has been the standard of care to treat B cell lymphoma for many years (Keating G M, BioDrugs—: clinical immunotherapeutics, biopharmaceuticals and gene therapy 2011, 25(1):55-61). The combination of cyclophosphamide, doxorubicin, vincristine, and prednisone, also known as CHOP, is commonly used and effective in combating non-Hodgkin's lymphoma (Cullen et al. The New England journal of medicine 2005, 353(10):988-998).
Cyclophosphamide is a nitrogen mustard-related compound that has, similar to chlorambucil and melphalan, been hypothesized to be a potential substrate for MRP1/Mrp1 once conjugated to glutathione (Zhang et al. Drug metabolism reviews 2005, 37(4):611-703). In fact, a correlation was observed between MRP1 expression and poor response to cyclophosphamide for breast cancer patients using this drug (Filipits et al. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 2005, 23(6):1161-1168). Vincristine and doxorubicin, two other compounds that are included in CHOP, are also known substrates for MRP1/Mrp1 (Abe et al. British journal of cancer 1995, 72(2):418-423; van Tellingen et al. British journal of cancer 2003, 89(9):1776-1782; de Cremoux et al. Pediatric blood & cancer 2007, 48(3):311-317; and Kang et al. Anticancer research 1997, 17(5A):3531-3536). Despite the fact that it has been known for some time that treatment with multiple cancer drugs is more efficacious than treatment with just one drug, and the impact can often be greater than an additive effect, the mechanism for such an interaction between treatments has remained elusive (Rubin P: Jama 1973, 223(2):164-166; Tomashefsky et al. Oncology 1964, 17:1-6; and Nicholson et al. British medical journal 1970, 3(5713):7-10). It has long been thought that the nitrogen mustard HN2 can sensitize tumors to other cancer chemotherapeutic agents simply by causing DNA damage (Cheson B D & Leoni L: Clinical advances in hematology & oncology: H&O 2011, 9(8 Suppl 19):1-11).
Glutathione-conjugated electrophiles are common substrates for MRP1/Mrp1. Glutathione-conjugated ethacrynic acid derivatives have been disclosed to inhibit MRP1 in the low micromolar range (Burg et al. Molecular pharmacology 2002, 62(5):1160-1166). Triazole-bridged dimers have also been disclosed to be effective inhibitors of MRP1 in the nanomolar (73 to 133) range (Wong et al. J Med Chem 2009, 52(17):5311-5322).